1. Field of the Invention
The invention relates to macrocyclic rare earth complexes, to a method of reducing perturbations in a fluorescent assay using these complexes, and to their use for reducing the perturbations in the measuring medium of a fluorescent method of detecting and/or determining an analyte in a medium in which it may be present.
2. Description of the Prior Art
Immunoassays are widely used at the present time for the qualitative and quantitative analysis of compounds in biological fluids.
Among the techniques which exist, fluorimetric assays have become increasingly important.
In fact, they have a number of advantages, including the sensitivity and rapidity of the measurement, the stability and safety of the reagents labeled with fluorescent compounds, and the relatively low cost.
It is known that methods of detection which use fluorescence are intrinsically very sensitive and might permit lower detection limits than those achieved by immunoassays which use radiolabeled reagents, in particular when using modulatable laser light sources (I. Wieder, Immunofluorescence and related staining techniques, 1978, Elsevier).
A large number of fluorescent molecules which can be used as tracers in assays of this type have been described previously and include rare earth complexes possessing valuable properties.
The use of particular complexes, namely rare earth cryptates, is described in the patent applications EP 0 180 492, PCT/FR86/00269, EP 0 321 353 or
These rare earth cryptates have the advantage of being very stable in a saline protein medium, this property being particularly important in the case of homogeneous immunoassays.
The sensitivity of the measurement can nevertheless be greatly affected by difference types of perturbation resulting from the presence of various molecules in the measuring medium.
This problem is particularly acute in the case of assays in a serum medium in which numerous molecules are capable of interfering.
For example, the measured signal can be perturbed by the emission of molecules capable of being excited and of emitting at the same wavelengths as the molecule used as the tracer.
The time-resolved methods of measuring fluorescence enable this disadvantage to be partially overcome. The principle of these methods consists in measuring the fluorescence emitted by a tracer molecule having a relatively long emission lifetime, the measurement being delayed in time beyond the emission lifetime of the other molecules present.
In this case it is necessary to use fluorescent tracer molecules with a relatively long lifetime, such as rare earth chelates.
The sensitivity of the measurement can also be affected by interference from molecules in the medium capable of perturbing the variation in fluorescence resulting from the bonding between the analyte to be detected and the labeled biospecific reagent. The patent application EP 0 324 323 describes the use of a modulator which stabilizes the rare earth chelate bonded to the biospecific reagent, so that the measured fluorescence is a true function of the concentration of the analyte. The effect of this modulator is to prevent perturbation of the fluorescence of the rare earth chelate by the other molecules present in the medium. The measured variation in fluorescence is then a function of the antigen-antibody reaction only. The proposed modulators are macromolecules such as proteins and detergents, and have to be used in excess in the range from 0.1 to 10 g/l.
Nevertheless, none of these methods completely solves the problem of the perturbations due to the molecules present in the measuring medium. In fact, an important source limiting the sensitivity of the fluorescent measurement is the existence of quenching processes due to molecules present in the medium which are capable of inhibiting the fluorescence of the fluorescent molecule used as the marker in the assay. In the case of rare earth complexes, these processes can result from proximity electron transfer mechanisms, in which the inhibitor molecule occupies the coordination sites remaining free within the complex. Particular mention may be made of the redox reactions occurring between the fluorescent molecule, in its ground state or in its excited state, and molecules present in the medium. These mechanisms are capable of causing a considerable variation in the emitted fluorescence.
The article by Weber et al., Clin. Chem., 1983, 29/9, 1665-1672, describes the influence of perturbations due in particular to uric acid in the amperometric detection of a tris(2,2'-bipyridine)ruthenium(III) complex. This complex is produced by the redox reaction of the corresponding Ru(II) complex, which is capable of oxidizing a Co(III) quenching complex. Uric acid has been identified as a reducing agent for Ru(III) and is therefore capable of interfering in the measurement.
This redox mechanism, due to an electron transfer between a fluorescent compound and a quenching compound, has also been demonstrated by Sabbatini et al., J.A.C.S., 1984, 106, 4055-4056. This article describes in particular the oxidation of a complex M(CN).sub.6.sup.4-, in which M is iron, ruthenium or osmium, by a europium cryptate.
The inhibition of fluorescence by mechanisms involving an electron transfer, and by quenching mechanisms in general, is an extremely troublesome phenomenon in practice because the inhibiting factors can either be naturally present as components in the measuring medium (for example uric acid in serum) or else be added thereto as additives or stabilizers for the assay.
These inhibitors greatly affect the fluorescence of the marker molecule. In particular, in the case of redox perturbing reactions, the conversion of a rare earth ion from the reduced state to the oxidized state via a redox mechanism results in a decrease in the lifetime and a modification of the emission spectrum of the complex in which it is present, thereby greatly affecting the sensitivity of the measurement.
The use of ligands of the macrocyclic type for chelating rare earth ions has been described in the literature, especially in the following publications: J. Phys. Chem., 1987, 91, 4681-4685, Inorg. Chem., 1983, 22, 3866-3869, Inorg. Chem. Acta, 1984, 95, 119-125, Nucl. Med. Biol., 1986, 13, 311-318, and Comprehensive Coordination Chemistry, 1987, vol. 3, published by Pergamon Press, and in the publication Helvetica Chimica Acta, 1990, 73, 1149-1162.
Although some of the compounds described have a low dissociation rate in water, the criterion of stability in pure water is not sufficient since serum actually contains numerous ions and proteins, some of them at high concentration, which can compete with the ligand for complexation of the rare earth ion.